The goal of this research is to elucidate, at the molecular level, novel mechanisms responsible for generating the consistent left-right (LR) asymmetry of the vertebrate body. We will test what role ion flux mechanisms play in establishing LR asymmetry when thousands of cells are present and identify what role the cytoskeleton plays in the asymmetric distribution of ion transporters. Recent work from the frog indicates that ion flux mechanisms are involved in early LR patterning. Specifically, four ion transporters are asymmetrically distributed across the embryo's midline during the first two cell cleavages and are required for proper LR asymmetry. These data present a clear picture of steps linking subcellular localization of ion transporters and downstream asymmetric patterning. However, this mechanism relies on the fact that in Xenopus, the cleavage plane of the first large blastomeres normally coincides with the prospective midline of the whole embryo. Surprisingly, when a second organizer is induced in frog embryos during the late blastula stage (~1000 cells), the ectopic twin has normal LR asymmetry. Unlike the large early blastomeres, individual cells at this stage cannot distribute components across the embryonic midline by intracellular localization;nonetheless correct laterally can be imposed upon the small cells of the late blastula - a context similar to mammalian LR initiation. Therefore, some mechanism must properly pattern embryos when thousands of small cells are present. We will test whether the mechanisms that operate in early frog embryos are re-used when tissues with thousands of cells are present. Two aims will be addressed: (1) To determine whether ion flux mechanisms are required to orient the normal asymmetry of the LR axis during late stage embryogenesis. (2) To understand how cytoskeletal motor proteins establish bioelectrical polarity in early and late embryos. In the first aim, we will induce ectopic twins at a stage when thousands of cells are present and large-scale, midline-oriented cleavages are not occurring in the frog embryo. We will then test four different ion flux mechanisms that have been implicated in the establishment of the LR axis during the first cleavages to determine whether any also function in later stages. In the second aim, we will disrupt individual motor proteins'function at early and late stages and assess the resulting effect on asymmetry of ion transporter localization, to shed light on intracellular transport on early and late LR initiation. Relevance: Laterality is a crucial aspect of embryonic development, particularly of morphogenesis of the cardiovascular system, visceral organs, and the brain. Understanding how asymmetry is established will help address birth defects and advance regenerative medicine of the heart and other organs.